The overall goal of our laboratory is to understand the mechanisms of selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) and to search for specific neuroprotective strategies applicable to motor neuron degenerations. The specific goal of this proposal is to determine whether (and by what mechanisms) recently engineered recombinant neurotrophic factors can be protective by (1) preserving the phenotype and survival of mature motor neurons, and (2) protecting them from experimentally induced chronic degeneration. Inevitably, much of the previous work identifying the role of neurotrophic factors in motor neuron survival has focused on models of the developing nervous system in which motor neurons undergo naturally occurring cell death (apoptosis). Few studies have actually considered the role of neurotrophic factors in postnatal motor neuron survival, when the process of naturally occurring motor neuron death is no longer applicable. The work outlined in this proposal will broaden our understanding of the effects of neurotrophic factors on motor neuron survival in the mature nervous system. Several such factors (CNTF, IGF) have already been rushed into clinical trials (some would say prematurely) for neurodegenerative diseases. This work will provide important preclinical data on the use of these factors as therapy in motor neuron diseases. A unique organotypic culture system has been developed in our laboratory for postnatal rat spinal cord. Because this preparation combines the advantages of long-term survival of mature motor neurons in culture with partially preserved synaptic connections, it is an ideal system in which to test whether neurotrophic factors can affect mature motor neuron survival or provide protection from degeneration. It simultaneously provides a way of determining any inherent toxicity of these growth factors, particularly those with cytokine like activity. The specific localization of choline acetyltransferase activity (ChAT) to large motor neurons in the ventral lumbar cord allows techniques for systematically assessing motor neuron viability. These include biochemical assay of ChAT activity and immunocytochemical staining of ChAT for morphologic analysis and cell counts. We also use the organotypic spinal cord cultures to provide a novel model of slow, glutamate-mediated motor neuron degeneration, a postulated mechanism of neurotoxicity in ALS. Long term incubation of spinal cord in the presence of threohydroxyaspartate, a potent glutamate transport inhibitor, reproduces relatively selective motor neuron degeneration. In this model, the neuroprotectant effects of the neurotrophic factors on motor neurons will be studied, providing further insight into the mechanisms of neurotrophic actions and potential pharmacologic strategies for therapies of motor neuron diseases such as ALS.